Fuel ethanol is currently produced from feedstocks such as corn starch, sugar cane, and sugar beets. However, the potential for production of ethanol from these sources is limited as most of the farmland which is suitable for the production of these crops is already in use to provide a food source for humans and animals. Furthermore, the production of ethanol from these feedstocks has significant greenhouse gas emissions because fossil fuels are used in the conversion process.
The production of ethanol from cellulose-containing feedstocks, such as agricultural residues, grasses, and forestry residues, has received much attention in recent years. The reasons for this are because these feedstocks are widely available and inexpensive and their use for ethanol production provides an alternative to burning or landfilling lignocellulosic waste materials. Moreover, a portion of the feedstock, lignin, can be used as a fuel to power the process, rather than fossil fuels. Several studies have concluded that, when the entire production and consumption cycle is taken into account, the use of ethanol produced from cellulose generates close to zero greenhouse gases.
The lignocellulosic feedstocks that are the most promising for ethanol production include (1) agricultural residues such as corn stover, wheat straw, barley straw, oat straw, rice straw, canola straw, and soybean stover; (2) grasses such as switch grass, miscanthus, cord grass, and reed canary grass; (3) fiber process residues such as corn fiber, beet pulp, pulp mill fines and rejects and sugar cane bagasse; (4) forestry wastes such as aspen wood, other hardwoods, softwood and sawdust; and (5) post-consumer waste paper products.
The most important process step of converting a lignocellulosic feedstock to ethanol involves converting the cellulose to glucose, for subsequent conversion to ethanol by fermentation. The two primary processes for accomplishing this are acid hydrolysis, which involves the hydrolysis of the feedstock using a single step of acid treatment, and enzymatic hydrolysis, which involves an acid pretreatment followed by hydrolysis with cellulase enzymes.
In the acid hydrolysis process, the feedstock is typically subjected to steam and sulfuric acid at a temperature, acid concentration and length of time that are sufficient to hydrolyze the cellulose to glucose and the hemicellulose to xylose and arabinose. The acid can be concentrated (25-80% w/w) or dilute (3-8% w/w). The glucose is then fermented to ethanol using yeast, and the ethanol is recovered and purified by distillation. Optionally, the glucose may be fermented to lactic acid, butanol, or other products.
In the enzymatic hydrolysis process, the reaction conditions are chosen such that the cellulose surface area is greatly increased as the fibrous feedstock is converted to a muddy texture, but there is little conversion of the cellulose to glucose. The pretreated cellulose is then hydrolyzed to glucose in a subsequent step that uses cellulase enzymes, and the steam and/or acid treatment in this case is known as pretreatment. The glucose may then be fermented to ethanol, lactic acid, butanol, or other products. Prior to the addition of enzyme, the pH of the acidic feedstock is adjusted to a value that is suitable for the enzymatic hydrolysis reaction. Typically, this involves the addition of alkali to a pH of between about 4 to about 6, which is the optimal pH range for many cellulases, although the pH can be higher if alkalophilic cellulases are used.
In one type of pretreatment process, the pressure produced by the steam is brought down rapidly with explosive decompression, which is known as steam explosion. Foody (U.S. Pat. No. 4,461,648) describes the equipment and conditions used in steam explosion pretreatment. Steam explosion with sulfuric acid carried out at a pH from 0.4 to 2.0 produces pretreated material that is uniform and requires less cellulase enzyme to hydrolyze cellulose than other pretreatment processes.
Cellulase enzymes catalyze the hydrolysis of the cellulose (hydrolysis of β-1,4-D-glucan linkages) in the feedstock into products such as glucose, cellobiose, and other cellooligosaccharides. Cellulase is a generic term denoting a multienzyme mixture comprising exo-acting cellobiohydrolases (CBHs), endoglucanases (EGs) and β-glucosidases (βG) that can be produced by a number of plants and microorganisms. The enzymes in the cellulase of Trichoderma reesei which contain a cellulose binding domain include CBH1 (more generally, Cel7A), CBH2 (Cel6A), EG1 (Cel7B), EG2 (Cel5), EG4 (Cel61A), EG5 (Cel45A), EG6 (Cel74A), Cip1, Cip2, acetyl xylan esterase, β-mannanase, and swollenin. EG3 (Cel12) is an example of a cellulolytic enzyme without a cellulose binding domain.
Cellulase enzymes work synergistically to hydrolyze cellulose to glucose. CBH1 and CBH2 act on opposing ends of cellulose chains to liberate cellobiose (Barr et al., 1996), while the endoglucanases act at internal locations in the cellulose. The primary product of these enzymes is cellobiose, which is further hydrolyzed to glucose by β-glucosidase. It is known that most CBHs and EGs bind to cellulose in the feedstock via carbohydrate-binding modules (CBMs), such as cellulose-binding domains (CBDs), while most β-glucosidase enzymes, including Trichoderma and Aspergillus β-glucosidase enzymes, do not contain such binding modules and thus remain in solution during hydrolysis. Cellulase enzymes may contain a linker region that connects the catalytic domain to the carbohydrate binding module. The linker region is believed to facilitate the activity of the catalytically active domain.
The kinetics of the enzymatic hydrolysis of insoluble cellulosic substrates by cellulases do not follow simple Michaelis-Menten behaviour (Zhang et al., 1999). Specifically, increasing the dosage of cellulase in a hydrolysis reaction does not provide a linearly dependent increase in the amount of glucose produced in a given time. There is also a significant decrease in the rate of reaction as cellulose hydrolysis proceeds (Tolan, 2002). Several explanations have been proposed to explain the decline in the reaction rate. The major hypotheses include product inhibition, increasing recalcitrance of substrate through the course of a hydrolysis and enzyme inactivation.
The kinetics of cellulase action make the enzymatic hydrolysis of pretreatment material an inefficient step in the production of cellulose ethanol. Reduction of the costs associated with enzymatic hydrolysis, through increasing cellulase production or activity, has been identified as a major opportunity for cost savings (Sheehan, 1999). A recent report from a cellulosic ethanol workshop sponsored by the United States Department of Energy estimated a 10- to 25-fold higher cost for producing the enzymes for cellulose ethanol than for the enzymes required to produce ethanol from starch (Houghton, 2006).
Several approaches have been taken to increase the activity of cellulase mixtures. Increasing the amount of β-glucosidase produced by the microorganism which also secretes the mixed cellulases alleviates product inhibition by cellobiose (U.S. Pat. No. 6,015,703). Rational design or random mutagenesis techniques can also be used to modulate the properties of individual enzymes, as demonstrated by the production of a CBH1 variant with increased thermostability (US2005/0277172). An alternate method to explore genetic diversity is to directly survey enzyme homologs from many cellulolytic species. This approach has been taken with CBH1 (US2004/0197890) and CBH2 (US2006/0053514). The construction of a fusion protein combining complementary activities from endo- and exo-cellulolytic enzymes has also been demonstrated (US2006/0057672). The modular domain structure of cellulolytic enzymes has permitted the construction of enzymes comprising the catalytic domain. The absence of a CBD has dramatic effects on the substrate binding and activities of these enzymes (US2004/0053373, US2006/0008885). Genetically engineered cellulose-hydrolyzing enzymes have been created that comprise novel combinations of the catalytically active domain, the linker region and the CBD (U.S. Pat. No. 5,763,254).
All of the approaches described above, while targeting different aspects of the enzymatic hydrolysis of cellulose, have not increased the cellulase activity sufficiently to overcome the high cost of cellulase for cellulose hydrolysis. One drawback of these strategies has been the focus on a single enzyme at a time, neglecting the synergies possible with other cellulolytic enzymes.
Therefore, a better approach to increasing the activity of a cellulase system is to focus on maximizing the activity of a mixture containing more than one cellulase component. It has been reported that the efficiency of cellulose hydrolysis by a combination of endo- and exo-cellulases is much greater than would be expected by summing the activities of these enzymes acting in isolation (Wood and McCrae, 1979). Previous studies have measured the synergy between the cellulolytic enzymes of T. reesei by observing the behaviour of binary or ternary mixtures (e.g. Nidetsky et al., 1994). For example, Wood et al. (1989) studied binary, tertiary and septenary blends of cellobiohydrolases and endoglucanases from Penicillium pinophilum. However, synergism was not observed in the binary blends of Penicillium enzymes. The synergism of binary combinations of three enzymes from the bacterium Thermobifida fusca, two of which are from the same families as those comprising the major Trichoderma enzymes, has also been characterized (Jeoh, 2006). However, improved performance has not been reported.
Several attempts have been made to develop a blend of CBHs and EGs to maximize the amount of cellulose hydrolysis for a given enzyme dosage (the enzyme dosage is the mass of enzyme required to hydrolyse a given mass of cellulose). For example, optimized blends of cellulases of mixed origin including bacteria, largely derived from T. fusca, have been reported (Irwin, 1993; Walker et al., 1993; Kim et al., 1998). Trichoderma CBH1 and CBH2 were included in these studies, but bacteria are unable to produce family 7 cellulases and EG1 was therefore not part of these blends. Baker et al. (1998) attempted to determine an optimal blend of Trichoderma CBH1, CBH2, and EG1, but did not include EG2 in their experiment. In the case of Baker et al., the substrate was Sigmacell, a microcrystalline cellulose preparation. Boisset et al. (2001) performed an optimization of a ternary blend of CBH1, CBH2 and EG5 derived from Humicola insolens on the substrate of bacterial cellulose. However, these studies have not succeeded in developing cellulase enzyme mixtures with improved performance for the hydrolysis of cellulose within pretreated lignocellulosic biomass.
Thus, in spite of much research effort, there remains a need for an improved cellulase enzyme mixture for the hydrolysis of cellulose in a pretreated lignocellulosic feedstock. The absence of such an enzyme mixture represents a large hurdle in the commercialization of cellulose conversion to soluble sugars including glucose for the production of ethanol and other products.